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. 2024 Mar 25;25(7):3643.
doi: 10.3390/ijms25073643.

Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies

Khondamir Imomnazarov  1 , Joshua Lopez-Scarim  1 , Ila Bagheri  1 , Valerie Joers  1 , Malú Gámez Tansey  1   2 , Alfonso Martín-Peña  1
Affiliations

Biochemical Fractionation of Human α-Synuclein in a Drosophila Model of Synucleinopathies

Khondamir Imomnazarov et al. Int J Mol Sci. .

Abstract

Synucleinopathies are a group of central nervous system pathologies that are characterized by the intracellular accumulation of misfolded and aggregated α-synuclein in proteinaceous depositions known as Lewy Bodies (LBs). The transition of α-synuclein from its physiological to pathological form has been associated with several post-translational modifications such as phosphorylation and an increasing degree of insolubility, which also correlate with disease progression in post-mortem specimens from human patients. Neuronal expression of α-synuclein in model organisms, including Drosophila melanogaster, has been a typical approach employed to study its physiological effects. Biochemical analysis of α-synuclein solubility via high-speed ultracentrifugation with buffers of increasing detergent strength offers a potent method for identification of α-synuclein biochemical properties and the associated pathology stage. Unfortunately, the development of a robust and reproducible method for the evaluation of human α-synuclein solubility isolated from Drosophila tissues has remained elusive. Here, we tested different detergents for their ability to solubilize human α-synuclein carrying the pathological mutation A53T from the brains of aged flies. We also assessed the effect of sonication on the solubility of human α-synuclein and optimized a protocol to discriminate the relative amounts of soluble/insoluble human α-synuclein from dopaminergic neurons of the Drosophila brain. Our data established that, using a 5% SDS buffer, the three-step protocol separates cytosolic soluble, detergent-soluble and insoluble proteins in three sequential fractions according to their chemical properties. This protocol shows that sonication breaks down α-synuclein insoluble complexes from the fly brain, making them soluble in the SDS buffer and thus enriching the detergent-soluble fraction of the protocol.

Keywords: Drosophila; Parkinson’s disease; SDS; chemical fractionation; synucleinopathy; α-synuclein.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article.

Figures

Figure 1
Figure 1
Human A53T α-synuclein extracted from Drosophila dopaminergic neurons exhibits higher solubility in RIPA than in the SDS buffer. (A) Schematic representation of the sequential fractionation protocol and the extraction buffers employed in this experiment. (B) Representative Western blot of head lysates from flies expressing hSNCAA53T in dopaminergic neurons. Fly heads were fractionated using a 3-step protocol in which the second fraction uses a variable detergent solvent, TBS, SDS or RIPA buffer. The first fraction (TBS-soluble) was loaded in lanes 1–4, second fraction (TBS-wash, SDS-soluble or RIPA-soluble) was loaded in lanes 5–8 and the third fraction (insoluble) was loaded in lanes 9–12, while 2 ng of purified recombinant human α-synuclein monomers (monomer) was loaded in lane 13 as positive control. Protein lysates were extracted from flies expressing hSNCAA53T in dopaminergic neurons (w; +/+; TH-Gal4/UAS-hSNCAA53T, lanes 1–3, 5–7, 9–11) and control flies (w; +/+; TH-Gal4/UAS-LacZ) as negative controls not expressing hSNCA (lanes 4, 8, 12). The fractions were probed for α-synuclein (4B12, top panel) and α-tubulin (T6074, bottom panel). (C) Quantification of α-synuclein content across independent experiments shows no significant differences between the different solvents in the TBS-soluble fraction (Tukey’s multiple comparisons; TBS vs. SDS, p = 0.9864; SDS vs. RIPA, p = 0.9972; TBS vs. RIPA, p = 0.9959) or the insoluble fraction (Tukey’s multiple comparisons; TBS vs. SDS, p = 0.7746; SDS vs. RIPA, p = 0.3137; TBS vs. RIPA, p = 0.1097) but significantly more α-synuclein was detected in the SDS- and RIPA-soluble fractions than in the TBS-wash (Tukey’s multiple comparisons; TBS vs. SDS, p = 0.0022; SDS vs. RIPA, p = 0.0048; TBS vs. RIPA, p < 0.0001). (D) Quantification of α-tubulin content shows no significant differences between the solvents (Tukey’s multiple comparisons; Fraction 1: TBS vs. SDS, p = 0.2462; SDS vs. RIPA, p = 0.4421; TBS vs. RIPA, p = 0.9298; Fraction 2: TBS vs. SDS, p = 0.6415; SDS vs. RIPA, p = 0.8869; TBS vs. RIPA, p = 0.9189; Fraction 3: TBS vs. SDS, p = 0.9736; SDS vs. RIPA, p = 0.9267; TBS vs. RIPA, p = 0.9899). Samples size was n = 4 (α-synuclein) and n = 3 (α-tubulin) for TBS samples, n = 5 (α-synuclein and α-tubulin) for SDS samples, and n = 5 (α-synuclein) and n = 3 (α-tubulin) for RIPA samples. Error bars indicate SD. Tukey’s comparison test: ns, not significant, ** p < 0.005, **** p < 0.0001.
Figure 2
Figure 2
Sonication increases solubility of human A53T α-synuclein in the SDS buffer. (A) Schematic representation of the sequential fractionation protocol and the extraction buffers employed in this experiment. (B) Representative Western blot of head lysates from flies expressing hSNCAA53T or LacZ in dopaminergic neurons. Fly heads were homogenized and then +/− sonicated prior to fractionation using a 3-step protocol in which the second fraction used SDS as the detergent solvent. The first fraction (TBS-soluble) was loaded in lanes 1, 2, 7, 8; the second fraction (SDS-soluble) was loaded in lanes 3, 4, 9, 10; and the third fraction (insoluble) was loaded in lanes 5, 6, 11, 12; while 2 ng of purified recombinant human α-synuclein monomers (monomer) was loaded in lane 13 as positive control. Protein lysates were extracted from flies expressing hSNCAA53T in dopaminergic neurons (w; +/+; TH-Gal4/UAS-hSNCAA53T, lanes 1, 3, 5, 7, 9, 11) and control flies (w; +/+; TH-Gal4/UAS-LacZ) as negative controls not expressing hSNCA (lanes 2, 4, 6, 8, 10, 12). The fractions were probed for α-synuclein (4B12, top panel) and α-tubulin (T6074, bottom panel). (C) Quantification of α-synuclein content shows no significant differences between sonicated and non-sonicated samples in the TBS- and SDS-soluble fractions but a significant increase in the amount of insoluble α-synuclein in the third, SDS-insoluble, fraction (Tukey’s multiple comparisons; TBS-soluble, p = 0.4969; SDS-soluble, p = 0.3459; SDS-insoluble, p = 0.0418). (D) Quantification of α-tubulin content shows no significant differences between sonication regimens (Tukey’s multiple comparisons; TBS-soluble, p = 0.3495; SDS-soluble, p = 0.9578; SDS-insoluble, p = 0.9398). Samples size was n = 5 for sonicated samples and n = 3 for non-sonicated samples. Error bars indicate SD. Tukey’s comparison test: ns, not significant, * p < 0.05.
Figure 3
Figure 3
Sonication does not affect human A53T α-synuclein solubility in RIPA buffer. (A) Schematic representation of the sequential fractionation protocol and the extraction buffers employed in this experiment. (B) Representative Eestern blot of head lysates from flies expressing hSNCAA53T or LacZ in dopaminergic neurons. Fly heads were homogenized and then +/− sonicated prior to fractionation using a 3-step protocol in which the second fraction uses RIPA buffer. The first fraction (TBS-soluble) was loaded in lanes 1, 2, 7, 8; the second fraction (RIPA-soluble) was loaded in lanes 3, 4, 9, 10; and the third fraction (insoluble) was loaded in lanes 5, 6, 11, 12; while 2 ng of purified recombinant human α-synuclein monomers (monomer) was loaded in lane 13 as positive control. Protein lysates were extracted from flies expressing hSNCAA53T in dopaminergic neurons (w; +/+; TH-Gal4/UAS-hSNCAA53T, lanes 1, 3, 5, 7, 9, 11) and control flies (w; +/+; TH-Gal4/UAS-LacZ) as negative controls not expressing hSNCA (lanes 2, 4, 6, 8, 10, 12). The fractions were probed for α-synuclein (4B12, top panel) and α-tubulin (T6074, bottom panel). (C) Quantification of α-synuclein content shows no significant differences between sonicated and non-sonicated samples in the any of the three fractions (Tukey’s multiple comparisons; TBS-soluble, p = 0.9803; RIPA-soluble, p = 0.9784; RIPA-insoluble, p > 0.9999). α-synuclein is not significantly detected in the RIPA-insoluble fraction (Student’s t-test from zero: sonicated, p = 0.2329; non-sonicated, p = 0.4226). (D) Quantification of α-tubulin content shows no significant differences between sonication regimens (Tukey’s multiple comparisons; TBS-soluble, p = 0.9995; RIPA-soluble, p > 0.9999; RIPA-insoluble, p = 0.9965). Samples size was n = 5 (α-synuclein) and n = 3 (α-tubulin) for sonicated samples, and n = 3 for non-sonicated samples. Error bars indicate SD. Tukey’s comparison test: ns, not significant.
Figure 4
Figure 4
Human A53T α-synuclein solubility in buffers containing the polyethoxylate detergent NP-40. (A) Schematic representation of the sequential fractionation protocol and the extraction buffers employed in this experiment. (B) Representative Western blot of head lysates from flies expressing hSNCAA53T or LacZ in dopaminergic neurons. Fly heads were homogenized and then +/− sonication prior to fractionation using a 3-step protocol in which the second fraction uses NP-40 as detergent solvent. The first fraction (TBS-soluble) was loaded in lanes 1, 2, 7, 8; the second fraction (NP-40-soluble) was loaded in lanes 3, 4, 9, 10; the third fraction (insoluble) was loaded in lanes 5, 6, 11, 12; and 2 ng of purified recombinant human α-synuclein monomers (monomer) was loaded in lane 13 as positive control. Protein lysates were extracted from flies expressing hSNCAA53T in dopaminergic neurons (w; +/+; TH-Gal4/UAS-hSNCAA53T, lanes 1, 3, 5, 7, 9, 11) and control flies (w; +/+; TH-Gal4/UAS-LacZ) as negative controls not expressing hSNCA (lanes 2, 4, 6, 8, 10, 12). The fractions were probed for α-synuclein (4B12, top panel) and α-tubulin (T6074, bottom panel). (C) Quantification of α-synuclein content shows no significant differences between sonicated and non-sonicated samples in the any of the three fractions (Tukey’s multiple comparisons, sonicated vs. non-sonicated: TBS-soluble, p = 0.9990; NP-40-soluble, p = 0.5587; NP-40-insoluble, p > 0.9999). α-synuclein is not significantly detected in the NP-40-insoluble fraction (Student’s t-test from zero: sonicated, p = 0.2292; non-sonicated, p = 0.2201). (D) Quantification of α-tubulin shows with no significant differences between sonication regimens (Tukey’s multiple comparisons, sonicated vs. non-sonicated: TBS-soluble, p = 0.2177; NP-40-soluble, p = 0.9994; NP-40-insoluble, p = 0.9741). Samples size was n = 3 for sonicated samples and n = 3 for non-sonicated samples. Error bars indicate SD. Tukey’s comparison test: ns, not significant.
Figure 5
Figure 5
Human A53T α-synuclein solubility in buffers containing the polyethoxylate detergent Triton X-100. (A) Schematic representation of the sequential fractionation protocol and the extraction buffers employed in this experiment. (B) Representative Western blot of head lysates from flies expressing hSNCAA53T or LacZ in dopaminergic neurons. Fly heads were homogenized and then +/− sonication prior to fractionation employing a 2-step protocol with Triton X-100 as detergent solvent. The first fraction (Triton X-100-soluble) was loaded in lanes 1, 2, 5, 6; the second fraction (Triton X-100-insoluble) was loaded in lanes 3, 4, 7, 8; and 2 ng of purified recombinant human α-synuclein monomers (monomer) was loaded in lane 9 as positive control. Protein lysates were extracted from flies expressing hSNCAA53T in dopaminergic neurons (w; +/+; TH-Gal4/UAS-hSNCAA53T, lanes 1, 3, 5, 7) and control flies (w; +/+; TH-Gal4/UAS-LacZ) as negative controls not expressing hSNCA (lanes 2, 4, 6, 8). The fractions were probed for α-synuclein (4B12, top panel) and α-tubulin (T6074, bottom panel). (C,D) Quantification of α-synuclein (C) and α-tubulin (D) content shows no significant differences between sonicated and non-sonicated samples in the any of the two fractions (Tukey’s multiple comparisons, sonicated vs. non-sonicated: For α-synuclein Triton-X-100-soluble, p = 0.3562; Triton-X-100-insoluble, p = 0.9998; For α-tubulin Triton-X-100-soluble, p = 0.6973; Triton-X-100-insoluble, p > 0.9999). α-synuclein is significantly detected in the Triton X-100-soluble fraction (Student’s t-test from zero: sonicated, p = 0.0019; non-sonicated, p = 0.0061) but not significantly in the Triton-X-100-insoluble fraction (Student’s t-test from zero: sonicated, p = 0.3910; non-sonicated, p = 0.4226). α-tubulin is significantly detected in the Triton X-100-soluble fraction (Student’s t-test from zero: sonicated, p = 0.0296; non-sonicated, p = 0.0162) and not detected in the Triton-X-100-insoluble fraction. Samples size was n = 4 (α-synuclein) and n = 3 (α-tubulin) for sonicated samples, and n = 3 for non-sonicated samples. Error bars indicate SD. Tukey’s comparison test: ns, not significant.
Figure 6
Figure 6
Chemical structures of the detergents NP-40, Triton X-100 and SDS employed in this study. (A) NP-40, polyethylene glycol nonyl-phenyl ether or nonoxynol-40. (B) Triton X-100, polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether, or octyl phenol ethoxylate. (C) Sodium dodecyl sulfate (SDS). Note the structural similarities between the polyethoxylated detergents, NP-40 and Triton X-100, with long chains of ethyl ether groups (24–51 for NP-40 and 9–10 for Triton X-100), which are lacking from the structure of SDS.
Figure 7
Figure 7
Schematic representation of the procedure for serial protein fractionation from Drosophila heads. (A) A 3-step sequential ultracentrifugation protocol for extracting insoluble α-synuclein from fly heads using TBS, SDS, RIPA or NP-40 buffers (see Table 1 for specific buffer composition). (B) A 2-step sequential ultracentrifugation protocol for extraction of α-synuclein from fly heads using Triton X-100 as a solvent in the fractionation buffer.
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